The phosphoenolpyruvate (PEP)-dependent carbohydrate:phosphotransferase system (PTS), a major sugar uptake system widely distributed among bacteria, plays key roles in various aspects of signaling, including chemotaxis. Enzyme I (EI), the first component of PTS, accepts a phosphoryl group from PEP, which is then transferred through a chain of proteins eventually to the substrate sugar. In chemotaxis, unphosphorylated EI inhibits the activity of the histidine kinase CheA, leading to an attractant response to the substrate sugar. In this study, I aimed at understanding the signaling connection between EI and CheA, whose mechanism has long been a mystery. EI fused with green fluorescent protein (GFP) showed polar and/or lateral dotted patterns of localization. The deletion of the cheA gene decreased localization of EI-GFP. However, the deletion of the MCP genes had a more severe effect and the presence of an MCP (Tar) in the absence of Che proteins supported the EI-GFP localization. Moreover, EI-GFP did not co-localize with RFP-CheA. Substitutions at the phosphorylation site of EI enhanced dotted localization but did not promote co-localization with CheA. These results cast a serious doubt on the direct interaction of EI with CheA that has been proposed in the literature. Overproduction of EI and EI-GFP in an effort to identify the target resulted in growth inhibition. Several spontaneous mutants with no growth inhibitory effect were isolated but the resulting variants of EI-GFP, though fluorescent, did not inhibit chemotaxis of the wild-type strain, suggesting that they lost the function to regulate chemotaxis. Thus, EI functions in cell growth and chemtotaxis cannot be dissected at present.